Therapeutic Polymeric Nanoparticles with mTOR Inhibitors and Methods of Making and Using Same

ABSTRACT

The present disclosure generally relates to therapeutic nanoparticles. Exemplary nanoparticles disclosed herein may include about 1 to about 20 weight percent of a mTOR inhibitor; and about 70 to about 99 weight percent biocompatible polymer.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/910,328 filedJun. 5, 2013, which is a continuation of U.S. Ser. No. 12/485,462 filedJun. 16, 2009, which claims priority to U.S. Ser. No. 61/061,760, filedJun. 16, 2008; U.S. Ser. No. 61/105,916, filed Oct. 16, 2008, U.S. Ser.No. 61/106,777, filed Oct. 20, 2008; U.S. Ser. No. 61/169,514, filedApr. 15, 2009; U.S. Ser. No. 61/175,209, filed May 4, 2009; U.S. Ser.No. 61/061,704, filed Jun. 16, 2008; U.S. Ser. No. 61/169,519, filedApr. 15, 2009; U.S. Ser. No. 61/175,219 filed May 4, 2009; U.S. Ser. No.61/061,697, filed Jun. 16, 2008; U.S. Ser. No. 61/088,159, filed Aug.12, 2008; U.S. Ser. No. 61/169,541, filed Apr. 15, 2009; U.S. Ser. No.61/175,226, filed May 4, 2009; U.S. Ser. No. 61/173,790, filed Apr. 29,2009; each of which is hereby incorporated by reference in theirentirety.

This invention was made with United States Government support underCooperative Agreement Number 70NANB7H7021 awarded by the NationalInstitute of Standard and Technology (NIST). The United StatesGovernment has certain rights in the Invention.

BACKGROUND

Systems that deliver certain drugs to a patient (e.g., targeted to aparticular tissue or cell type or targeted to a specific diseased tissuebut not normal tissue), or that control release of drugs has long beenrecognized as beneficial. For example, therapeutics that include anactive drug and that are capable of locating in a particular tissue orcell type e.g., a specific diseased tissue, may reduce the amount of thedrug in tissues of the body that do not require treatment. This isparticularly important when treating a condition such as cancer where itis desirable that a cytotoxic dose of the drug is delivered to cancercells without killing the surrounding non-cancerous tissue. Further,such therapeutics may reduce the undesirable and sometimes lifethreatening side effects common in anticancer therapy. For example,nanoparticle therapeutics may, due the small size, evade recognitionwithin the body allowing for targeted and controlled delivery whilee.g., remaining stable for an effective amount of time.

Therapeutics that offer such therapy and/or controlled release and/ortargeted therapy also must be able to deliver an effective amount ofdrug. It can be a challenge to prepare nanoparticle systems that have anappropriate amount of drug associated each nanoparticle, while keepingthe size of the nanoparticles small enough to have advantageous deliveryproperties. For example, while it is desirable to load a nanoparticlewith a high quantity of therapeutic agent, nanoparticle preparationsthat use a drug load that is too high will result in nanoparticles thatare too large for practical therapeutic use. Further, it may bedesirable for therapeutic nanoparticles to remain stable so as to e.g.substantially limit rapid or immediate release of the therapeutic agent.

Accordingly, a need exists for new nanoparticle formulations and methodsof making such nanoparticles and compositions, that can delivertherapeutic levels of drugs to treat diseases such as cancer, while alsoreducing patient side effects.

SUMMARY

In one aspect, the invention provides therapeutic nanoparticle thatincludes an active agent or therapeutic agent, e.g. an mTOR inhibitor orpharmaceutically acceptable salts thereof, and one, two, or threebiocompatible polymers. For example, disclosed herein is a therapeuticnanoparticle comprising about 1 to about 20 weight percent of atherapeutic agent (such as for example sirolimus, temsirolimus, oreverolimus) and about 50 to about 99 weight percent of a biocompatiblepolymer, e.g. about 70 to about 99 weight percent of a biocompatiblepolymer. For example, the biocompatible polymer may be a diblockpoly(lactic) acid-poly(ethylene)glycol copolymer (e.g. PLA-PEG) or adiblock (poly(lactic)-co-poly (glycolic) acid)-poly(ethylene)glycolcopolymer (e.g. PLGA-PEG), or the biocompatible polymer may two or morebiocompatible polymers, for example, the therapeutic nanoparticles canalso include a homopolymer such as poly(lactic) acid homopolymer. Forexample, a disclosed therapeutic nanoparticle may include about 1 toabout 20 weight percent, e.g about 2 to about 20 weight percent or about10 to about 20 weight percent, of a mTOR inhibitor; and about 70 toabout 99 weight percent biocompatible polymer, wherein the biocompatiblepolymer is selected from the group consisting of a) a diblockpoly(lactic) acid-poly(ethylene)glycol copolymer, b) a diblockpoly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol copolymer, c)a combination of a) or b) and a poly (lactic) acid homopolymer orpoly(lactic)-co-(glycolic) acid ; and d) a combination of a) or b) and apoly (lactic) acid homopolymer or poly(lactic)-co-(glycolic) acid.

The diameter of disclosed nanoparticles may be, for example, about 60 toabout 120 nm, or about 70 to about 120 nm. Disclosed therapeuticnanoparticles may be stable for at least 5 days at 25° C., e.g. mayremain stable over 5 days in vitro, e.g. in a sucrose solution. Inanother embodiment, disclosed particles may substantially immediatelyrelease less than about 2% or less than about 5%, or even less thanabout 10% of the therapeutic agent when placed in a phosphate buffersolution at room temperature, or at 37° C. For example, disclosednanoparticles may substantially retain the therapeutic agent for atleast 5 days at 25° C. In another embodiment, disclosed nanoparticlesmay release the therapeutic agent over a period of at least 1 day ormore when administered to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is flow chart for an emulsion process for forming disclosednanoparticle.

FIGS. 2A and 2B are flow diagrams for a disclosed emulsion process.

FIG. 3 depicts the effect of coarse emulsion preparation on quenchedparticle size. Placebo organic at 30% solids was used, emulsified at 5:1W:O using standard aqueous phase (1% sodium cholate, 2% benzyl alcohol,4% ethyl acetate).

FIG. 4 depicts the effect of feed pressure on resultant particle size.

FIG. 5 depicts the particle size dependence on scale. Placebo organicphase consisted of 25.5% polymer stock of 50:50 16.5/5 PLA/PEG:8.2 PLA.Organic phase was emulsified 5:1 O:W with standard aqueous phase, andmultiple discreet passes were performed, quenching a small portion ofemulsion after each pass. The indicated scale represents the totalsolids of the formulation.

FIG. 6 depicts the effect of solids concentration on particle size.

FIG. 7 depicts the effect of solids concentration and poly(lactic)homopolymer on loading percentage of sirolimus (rapamycin).

FIG. 8 depicts in vitro release of sirolimus over time for disclosednanoparticles.

FIG. 9 depicts the effects of poly(lactic) homopolymer on loadingpercentage of temsirolimus.

FIG. 10 depicts the effect of solids concentration on particle size oftemsirolimus containing particles.

FIG. 11 depicts in vitro release of temsirolimus over time for disclosednanoparticles

DETAILED DESCRIPTION

The present invention generally relates to polymeric nanoparticles thatinclude an active or therapeutic agent or drug, and methods of makingand using such therapeutic nanoparticles. In general, a “nanoparticle”refers to any particle having a diameter of less than 1000 nm, e.g.about 10 nm to about 200 nm. Disclosed therapeutic nanoparticles mayinclude nanoparticles having a diameter of about 60 to about 120 nm, orabout 70 to about 130 nm, or about 60 to about 140 nm, or about 70 nm toabout 140 nm.

Disclosed nanoparticles may include about 0.2 to about 35 weightpercent, about 3 to about 40 weight percent, about 5 to about 30 weightpercent, about 1 to about 20 weight percent, about 10 to about 30 weightpercent, about 5 to about 15 percent, about 15 to 25 weight percent, oreven about 4 to about 25 weight percent, e.g. about 10 weight percent ofan active agent, such as antineoplastic agent, e.g. a mTOR inhibitingagent (for example sirolimus, temsirolimus or everolimus).

Nanoparticles disclosed herein include one, two, three or morebiocompatible and/or biodegradable polymers. For example, a contemplatednanoparticle may include about 60 to about 99 weight percent of one,two, three or more biocompatible polymers such as one or moreco-polymers (e.g. a diblock polymer) that include a biodegradablepolymer (for example poly(lactic)acid and polyethylene glycol, andoptionally about 0 to about 50 weight percent of a homopolymer, e.g.biodegradable polymer such as poly(lactic) acid.

Polymers

In some embodiments, disclosed nanoparticles include a matrix ofpolymers. Disclosed nanoparticles may include one or more polymers, e.g.a diblock co-polymer and/or a monopolymer. Disclosed therapeuticnanoparticles include a therapeutic agent can may associated with thesurface of, encapsulated within, surrounded by, and/or dispersedthroughout a polymeric matrix.

A wide variety of polymers and methods for forming particles therefromare known in the art of drug delivery. In some embodiments, thedisclosure is directed toward nanoparticles with at least one polymer,for example, a first polymer that may be a co-polymer, e.g. a diblockco-polymer, and optionally a polymer that may be for example ahomopolymer.

Any polymer can be used in accordance with the present invention.Polymers can be natural or unnatural (synthetic) polymers. Polymers canbe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers can be random, block, or comprise a combinationof random and block sequences. Contemplated polymers may bebiocompatible and/or biodegradable.

The term “polymer,” as used herein, is given its ordinary meaning asused in the art, i.e., a molecular structure comprising one or morerepeat units (monomers), connected by covalent bonds. The repeat unitsmay all be identical, or in some cases, there may be more than one typeof repeat unit present within the polymer. In some cases, the polymercan be biologically derived, i.e., a biopolymer. Non-limiting examplesinclude peptides or proteins. In some cases, additional moieties mayalso be present in the polymer, for example biological moieties such asthose described below. If more than one type of repeat unit is presentwithin the polymer, then the polymer is said to be a “copolymer.” It isto be understood that in any embodiment employing a polymer, the polymerbeing employed may be a copolymer in some cases. The repeat unitsforming the copolymer may be arranged in any fashion. For example, therepeat units may be arranged in a random order, in an alternating order,or as a block copolymer, i.e., comprising one or more regions eachcomprising a first repeat unit (e.g., a first block), and one or moreregions each comprising a second repeat unit (e.g., a second block),etc. Block copolymers may have two (a diblock copolymer), three (atriblock copolymer), or more numbers of distinct blocks.

Disclosed particles can include copolymers, which, in some embodiments,describes two or more polymers (such as those described herein) thathave been associated with each other, usually by covalent bonding of thetwo or more polymers together. Thus, a copolymer may comprise a firstpolymer and a second polymer, which have been conjugated together toform a block copolymer where the first polymer can be a first block ofthe block copolymer and the second polymer can be a second block of theblock copolymer. Of course, those of ordinary skill in the art willunderstand that a block copolymer may, in some cases, contain multipleblocks of polymer, and that a “block copolymer,” as used herein, is notlimited to only block copolymers having only a single first block and asingle second block. For instance, a block copolymer may comprise afirst block comprising a first polymer, a second block comprising asecond polymer, and a third block comprising a third polymer or thefirst polymer, etc. In some cases, block copolymers can contain anynumber of first blocks of a first polymer and second blocks of a secondpolymer (and in certain cases, third blocks, fourth blocks, etc.). Inaddition, it should be noted that block copolymers can also be formed,in some instances, from other block copolymers. For example, a firstblock copolymer may be conjugated to another polymer (which may be ahomopolymer, a biopolymer, another block copolymer, etc.), to form a newblock copolymer containing multiple types of blocks, and/or to othermoieties (e.g., to non-polymeric moieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) can be amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer can be onegenerally that attracts water and a hydrophobic polymer can be one thatgenerally repels water. A hydrophilic or a hydrophobic polymer can beidentified, for example, by preparing a sample of the polymer andmeasuring its contact angle with water (typically, the polymer will havea contact angle of less than 60°, while a hydrophobic polymer will havea contact angle of greater than about 60°). In some cases, thehydrophilicity of two or more polymers may be measured relative to eachother, i.e., a first polymer may be more hydrophilic than a secondpolymer. For instance, the first polymer may have a smaller contactangle than the second polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) contemplated herein includes a biocompatible polymer, i.e.,the polymer that does not typically induce an adverse response wheninserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. Accordingly, thetherapeutic particles contemplated herein can be non-immunogenic. Theterm non-immunogenic as used herein refers to endogenous growth factorin its native state which normally elicits no, or only minimal levelsof, circulating antibodies, T-cells, or reactive immune cells, and whichnormally does not elicit in the individual an immune response againstitself.

Biocompatibility typically refers to the acute rejection of material byat least a portion of the immune system, i.e., a nonbiocompatiblematerial implanted into a subject provokes an immune response in thesubject that can be severe enough such that the rejection of thematerial by the immune system cannot be adequately controlled, and oftenis of a degree such that the material must be removed from the subject.One simple test to determine biocompatibility can be to expose a polymerto cells in vitro; biocompatible polymers are polymers that typicallywill not result in significant cell death at moderate concentrations,e.g., at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodiments ofthe present invention include polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, polycaprolactone, or copolymers or derivativesincluding these and/or other polymers.

In certain embodiments, contemplated biocompatible polymers may bebiodegradable, i.e., the polymer is able to degrade, chemically and/orbiologically, within a physiological environment, such as within thebody. As used herein, “biodegradable” polymers are those that, whenintroduced into cells, are broken down by the cellular machinery(biologically degradable) and/or by a chemical process, such ashydrolysis, (chemically degradable) into components that the cells caneither reuse or dispose of without significant toxic effect on thecells. In one embodiment, the biodegradable polymer and theirdegradation byproducts can be biocompatible.

For instance, a contemplated polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), thepolymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer can be degradedinto monomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymersmay be biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymers may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (for example, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof. In some embodiments, polyestersinclude, for example, polyanhydrides, poly(ortho ester) PEGylatedpoly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone),polylysine, PEGylated polylysine, poly(ethylene imine), PEGylatedpoly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA can be characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid-glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention can be characterized by a lacticacid:glycolic acid molar ratio of approximately 85:15, approximately75:25, approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, the ratio of lactic acid to glycolic acid monomersin the polymer of the particle (e.g., the PLGA block copolymer orPLGA-PEG block copolymer), may be selected to optimize for variousparameters such as water uptake, therapeutic agent release and/orpolymer degradation kinetics can be optimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, amino alkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully-polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine), polyethylene imine(PEI), and poly(amidoamine) dendrimers are contemplated for use, in someembodiments, in a disclosed particle.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains. Examples of these polyesters includepoly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester),. A polymer (e.g., copolymer, e.g.,block copolymer) containing poly(ethylene glycol) repeat units can alsobe referred to as a “PEGylated” polymer. Such polymers can controlinflammation and/or immunogenicity (i.e., the ability to provoke animmune response) and/or lower the rate of clearance from the circulatorysystem via the reticuloendothelial system (RES), due to the presence ofthe poly(ethylene glycol) groups.

PEGylation may also be used, in some cases, to decrease chargeinteraction between a polymer and a biological moiety, e.g., by creatinga hydrophilic layer on the surface of the polymer, which may shield thepolymer from interacting with the biological moiety. In some cases, theaddition of poly(ethylene glycol) repeat units may increase plasmahalf-life of the polymer (e.g., copolymer, e.g., block copolymer), forinstance, by decreasing the uptake of the polymer by the phagocyticsystem while decreasing transfection/uptake efficiency by cells. Thoseof ordinary skill in the art will know of methods and techniques forPEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminating inan amine, by ring opening polymerization techniques (ROMP), or the like.

Particles disclosed herein may or may not contain PEG. In addition,certain embodiments can be directed towards copolymers containingpoly(ester-ether)s, e.g., polymers having repeat units joined by esterbonds (e.g., R—C(O)—O—R′ bonds) and ether bonds (e.g., R—O—R′ bonds). Insome embodiments of the invention, a biodegradable polymer, such as ahydrolyzable polymer, containing carboxylic acid groups, may beconjugated with poly(ethylene glycol) repeat units to form apoly(ester-ether).

It is contemplated that PEG may include a terminal end group, forexample, when PEG is not conjugated to a ligand. For example, PEG mayterminate in a hydroxyl, a methoxy or other alkoxyl group, a methyl orother alkyl group, an aryl group, a carboxylic acid, an amine, an amide,an acetyl group, a guanidino group, or an imidazole. Other contemplatedend groups include azide, alkyne, maleimide, aldehyde, hydrazide,hydroxylamine, alkoxyamine, or thiol moieties.

In one embodiment, the molecular weight of the polymers can be optimizedfor effective treatment as disclosed herein. For example, the molecularweight of a polymer may influence particle degradation rate (such aswhen the molecular weight of a biodegradable polymer can be adjusted),solubility, water uptake, and drug release kinetics. For example, themolecular weight of the polymer can be adjusted such that the particlebiodegrades in the subject being treated within a reasonable period oftime (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8weeks, etc.). A disclosed particle can for example comprise a copolymerof PEG and PLGA, the PEG can have a molecular weight of 1,000-20,000,e.g., 5,000-20,000, e.g., 10,000-20,000, and the PLGA can have amolecular weight of 5,000-100,000, e.g., 20,000-70,000, e.g.,20,000-50,000.

For example, disclosed here is an exemplary therapeutic nanoparticlethat includes about 10 to about 99 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic)acid-poly(ethylene)glycol copolymer, or about 20 to about 80 weightpercent, about 40 to about 80 weight percent, or about 30 to about 50weight percent, or about 70 to about 90 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic)acid-poly(ethylene)glycol copolymer. Exemplary poly(lactic)acid-poly(ethylene)glycol copolymers can include a number averagemolecular weight of about 15 to about 20 kDa, or about 10 to about 25kDa of poly(lactic) acid and a number average molecular weight of about4 to about 6, or about 2 kDa to about 10 kDa of poly(ethylene)glycol.

Disclosed nanoparticles may optionally include about 1 to about 50weight percent poly(lactic) acid or poly(lactic) acid-co-poly (glycolic)acid (which does not include PEG, e.g a homopolymer of PLA), or mayoptionally include about 1 to about 50 weight percent, or about 10 toabout 50 weight percent or about 30 to about 50 weight percentpoly(lactic) acid or poly(lactic) acid-co-poly (glycolic) acid. Forexample, poly(lactic) or poly(lactic)-co-poly(glycolic) acid may have anumber average molecule weight of about 5 to about 15 kDa, or about 5 toabout 12 kDa. Exemplary homopolymeric PLA may have a number averagemolecular weight of about 5 to about 10 kDa. Exemplary PLGA may have anumber average molecular weight of about 8 to about 12 kDa.

In certain embodiments, disclosed polymers of may be conjugated to alipid, e.g. “end-capped,” for example, may include a lipid-terminatedPEG. As described below, the lipid portion of the polymer can be usedfor self assembly with another polymer, facilitating the formation of ananoparticle. For example, a hydrophilic polymer could be conjugated toa lipid that will self assemble with a hydrophobic polymer.

Exemplary lipids include fatty acids such as long chain (e.g., C₈-C₅₀),substituted or unsubstituted hydrocarbons. In some embodiments, a fattyacid group can be a C₁₀-C₂₀ fatty acid or salt thereof. In someembodiments, a fatty acid group can be a C₁₅-C₂₀ fatty acid or saltthereof. In some embodiments, a fatty acid can be unsaturated,monounsaturated, or polyunsaturated. For example, a fatty acid group canbe one or more of butyric, caproic, caprylic, capric, lauric, myristic,palmitic, stearic, arachidic, behenic, or lignoceric acid. In someembodiments, a fatty acid group can be one or more of palmitoleic,oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic, arachidonic,gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucicacid.

In a particular embodiment, the lipid is of the Formula V:

and salts thereof, wherein each R is, independently, C₁₋₃₀ alkyl. In oneembodiment of Formula V, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt.

In one embodiment, optional small molecule targeting moieties arebonded, e.g., covalently bonded, to the lipid component of thenanoparticle. For example, contemplated herein is also a nanoparticlecomprising a therapeutic agent, a polymeric matrix comprisingfunctionalized and non-functionalized polymers, a lipid, and alow-molecular weight targeting ligand, wherein the targeting ligand isbonded, e.g., covalently bonded, to the lipid component of thenanoparticle.

Targeting Moieties

Provided herein are nanoparticles that may include an optional targetingmoiety, i.e., a moiety able to bind to or otherwise associate with abiological entity, for example, a membrane component, a cell surfacereceptor, prostate specific membrane antigen, or the like. A targetingmoiety present on the surface of the particle may allow the particle tobecome localized at a particular targeting site, for instance, a tumor,a disease site, a tissue, an organ, a type of cell, etc. The drug orother payload may then, in some cases, be released from the particle andallowed to interact locally with the particular targeting site.

In one embodiment of the instant invention, the targeting moiety may bea low-molecular weight ligand, e.g., a low-molecular weight PSMA ligand.For example, a targeting portion may cause the particles to becomelocalized to a tumor, a disease site, a tissue, an organ, a type ofcell, etc. within the body of a subject, depending on the targetingmoiety used. For example, a low-molecular weight PSMA ligand may becomelocalized to prostate cancer cells. The subject may be a human ornon-human animal. Examples of subjects include, but are not limited to,a mammal such as a dog, a cat, a horse, a donkey, a rabbit, a cow, apig, a sheep, a goat, a rat, a mouse, a guinea pig, a hamster, aprimate, a human or the like.

Contemplated targeting moieties include small molecules. In certainembodiments, the term “small molecule” refers to organic compounds,whether naturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Small molecules typically havemultiple carbon-carbon bonds. In certain embodiments, small moleculesare less than about 2000 g/mol in size. In some embodiments, smallmolecules are less than about 1500 g/mol or less than about 1000 g/mol.In some embodiments, small molecules are less than about 800 g/mol orless than about 500 g/mol, for example about 100 g/mol to about 600g/mol, or about 200 g/mol to about 500 g/mol. For example, a ligand maybe a the low-molecular weight PSMA ligand such as

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof.

In some embodiments, small molecule targeting moieties that may be usedto target cells associated with prostate cancer tumors include PSMApeptidase inhibitors such as 2-PMPA, GPI5232, VA-033,phenylalkylphosphonamidates and/or analogs and derivatives thereof. Insome embodiments, small molecule targeting moieties that may be used totarget cells associated with prostate cancer tumors include thiol andindole thiol derivatives, such as 2-MPPA and3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives. In someembodiments, small molecule targeting moieties that may be used totarget cells associated with prostate cancer tumors include hydroxamatederivatives. In some embodiments, small molecule targeting moieties thatmay be used to target cells associated with prostate cancer tumorsinclude PBDA- and urea-based inhibitors, such as ZJ 43, ZJ 11, ZJ 17, ZJ38 and/or and analogs and derivatives thereof, androgen receptortargeting agents (ARTAs), polyamines, such as putrescine, spermine, andspermidine, inhibitors of the enzyme glutamate carboxylase II (GCPII),also known as NAAG Peptidase or NAALADase.

In another embodiment of the instant invention, the targeting moiety canbe a ligand that targets Her2, EGFR, or toll receptors. For example,contemplated the targeting moieties may include a nucleic acid,polypeptide, glycoprotein, carbohydrate, or lipid. For example, atargeting moiety can be a nucleic acid targeting moiety (e.g. anaptamer, e.g., the A10 aptamer) that binds to a cell type specificmarker. In general, an aptamer is an oligonucleotide (e.g., DNA, RNA, oran analog or derivative thereof) that binds to a particular target, suchas a polypeptide. In some embodiments, a targeting moiety may be anaturally occurring or synthetic ligand for a cell surface receptor,e.g., a growth factor, hormone, LDL, transferrin, etc. A targetingmoiety can be an antibody, which term is intended to include antibodyfragments, characteristic portions of antibodies, single chain targetingmoieties can be identified, e.g., using procedures such as phagedisplay. Targeting moieties may be a targeting peptide or targetingpeptidomimetic has a length of up to about 50 residues. For example, atargeting moieties may include the amino acid sequence AKERC, CREKA,ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids, orconservative variants or peptidomimetics thereof. In particularembodiments, the targeting moiety is a peptide that includes the aminoacid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z arevariable amino acids, and has a length of less than 20, 50 or 100residues. The CREKA (Cys Arg Glu Lys Ala) peptide or a peptidomimeticthereof peptide or the octapeptide AXYLZZLN are also contemplated astargeting moieties, as well as peptides, or conservative variants orpeptidomimetics thereof, that binds or forms a complex with collagen IV,or the targets tissue basement membrane (e.g., the basement membrane ofa blood vessel), can be used as a targeting moiety.

Exemplary targeting moieties include peptides that target ICAM(intercellular adhesion molecule, e.g. ICAM-1).

Targeting moieties disclosed herein are typically conjugated to adisclosed polymer or copolymer (e.g. PLA-PEG), and such a polymerconjugate may form part of a disclosed nanoparticle. For example, adisclosed therapeutic nanoparticle may optionally include about 0.2 toabout 10 weight percent of a PLA-PEG or PLGA-PEG, wherein the PEG isfunctionalized with a targeting ligand. Contemplated therapeuticnanoparticles may include, for example, about 0.2 to about 10 molepercent PLA-PEG-ligand or poly (lactic) acid -co poly (glycolic)acid-PEG-ligand. For example, PLA-PEG-ligand may include a PLA with anumber average molecular weight of about 10 kDa to about 20 kDa and PEGwith a number average molecular weight of about 4,000 to about 8,000.

Nanoparticles

Disclosed nanoparticles may have a substantially spherical (i.e., theparticles generally appear to be spherical), or non-sphericalconfiguration. For instance, the particles, upon swelling or shrinkage,may adopt a non-spherical configuration. In some cases, the particlesmay include polymeric blends. For instance, a polymer blend may includea first co-polymer that includes polyethylene glycol and a secondpolymer.

Disclosed nanoparticles may have a characteristic dimension of less thanabout 1 micrometer, where the characteristic dimension of a particle isthe diameter of a perfect sphere having the same volume as the particle.For example, the particle can have a characteristic dimension of theparticle can be less than about 300 nm, less than about 200 nm, lessthan about 150 nm, less than about 100 nm, less than about 50 nm, lessthan about 30 nm, less than about 10 nm, less than about 3 nm, or lessthan about 1 nm in some cases. In particular embodiments, disclosednanoparticles may have a diameter of about 70 nm-200 nm, or about 70 nmto about 180 nm, about 80 nm to about 130 nm, about 80 nm to about 120nm.

In one set of embodiments, the particles can have an interior and asurface, where the surface has a composition different from theinterior, i.e., there may be at least one compound present in theinterior but not present on the surface (or vice versa), and/or at leastone compound is present in the interior and on the surface at differingconcentrations. For example, in one embodiment, a compound, such as atargeting moiety (i.e., a low-molecular weight ligand) of a polymericconjugate of the present invention, may be present in both the interiorand the surface of the particle, but at a higher concentration on thesurface than in the interior of the particle, although in some cases,the concentration in the interior of the particle may be essentiallynonzero, i.e., there is a detectable amount of the compound present inthe interior of the particle.

In some cases, the interior of the particle is more hydrophobic than thesurface of the particle. For instance, the interior of the particle maybe relatively hydrophobic with respect to the surface of the particle,and a drug or other payload may be hydrophobic, and readily associateswith the relatively hydrophobic center of the particle. The drug orother payload can thus be contained within the interior of the particle,which can shelter it from the external environment surrounding theparticle (or vice versa). For instance, a drug or other payloadcontained within a particle administered to a subject will be protectedfrom a subject's body, and the body may also be substantially isolatedfrom the drug for at least a period of time.

For example, disclosed herein is a therapeutic polymeric nanoparticlecomprising a first non-functionalized polymer; an optional secondnon-functionalized polymer; an optional functionalized polymercomprising a targeting moiety; and a therapeutic agent, In a particularembodiment, the first non-functionalized polymer is PLA, PLGA, or PEG,or copolymers thereof, e.g. a diblock co-polymer PLA-PEG. For example,exemplary nanoparticle may have a PEG corona with a density of about0.065 g/cm³, or about 0.01 to about 0.10 g/cm³.

Disclosed nanoparticles may be stable (e.g. retain substantially allactive agent) for example in a solution that may contain a saccharide,for at least about 3 days, about 4 days or at least about 5 days at roomtemperature, or at 25° C.

In some embodiments, disclosed nanoparticles may also include a fattyalcohol, which may increase the rate of drug release. For example,disclosed nanoparticles may include a C₈-C₃₀ alcohol such as cetylalcohol, octanol, stearyl alcohol, arachidyl alcohol, docosonal, oroctasonal.

Nanoparticles may have controlled release properties, e.g., may becapable of delivering an amount of active agent to a patient, e.g., tospecific site in a patient, over an extended period of time, e.g. over 1day, 1 week, or more. In some embodiments, disclosed nanoparticlessubstantially immediately releases (e.g. over about 1 minute to about 30minutes) less than about 2%, less than about 4%, less than about 5%, orless than about 10% of an active agent (e.g. a taxane) agent, forexample when places in a phosphate buffer solution at room temperatureand/or at 37° C.

In one embodiment, the invention comprises a nanoparticle comprising 1)a polymeric matrix and 2) an amphiphilic compound or layer thatsurrounds or is dispersed within the polymeric matrix forming acontinuous or discontinuous shell for the particle, An amphiphilic layercan reduce water penetration into the nanoparticle, thereby enhancingdrug encapsulation efficiency and slowing drug release. Further, theseamphipilic layer protected nanoparticles can provide therapeuticadvantages by releasing the encapsulated drug and polymer at appropriatetimes.

As used herein, the term “amphiphilic” refers to a property where amolecule has both a polar portion and a non-polar portion. Often, anamphiphilic compound has a polar head attached to a long hydrophobictail. In some embodiments, the polar portion is soluble in water, whilethe non-polar portion is insoluble in water. In addition, the polarportion may have either a formal positive charge, or a formal negativecharge. Alternatively, the polar portion may have both a formal positiveand a negative charge, and be a zwitterion or inner salt. Exemplaryamphiphilic compound include, for example, one or a plurality of thefollowing: naturally derived lipids, surfactants, or synthesizedcompounds with both hydrophilic and hydrophobic moieties.

Specific examples of amphiphilic compounds include, but are not limitedto, phospholipids, such as 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine(DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratioof between 0.01-60 (weight lipid/w polymer), most preferably between0.1-30 (weight lipid/w polymer). Phospholipids which may be usedinclude, but are not limited to, phosphatidic acids, phosphatidylcholines with both saturated and unsaturated lipids, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines,phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, andβ-acyl-y-alkyl phospholipids. Examples of phospholipids include, but arenot limited to, phosphatidylcholines such asdioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcho-line (DBPC), ditricosanoylphosphatidylcholine(DTPC), dilignoceroylphatidylcholine (DLPC); andphosphatidylethanolamines such as dioleoylphosphatidylethanolamine or1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Syntheticphospholipids with asymmetric acyl chains (e.g., with one acyl chain of6 carbons and another acyl chain of 12 carbons) may also be used.

In a particular embodiment, an amphiphilic component may includelecithin, and/or in particular, phosphatidylcholine.

Preparation of Nanoparticles

Another aspect of the invention is directed to systems and methods ofmaking disclosed nanoparticles. In some embodiments, using two or moredifferent polymers (e.g., a copolymer such as a diblock copolymer and ahomopolymer) properties of particles may be controlled.

In a particular embodiment, the methods described herein formnanoparticles that have a high amount of encapsulated therapeutic agent,for example, may include about 1 to about 40 weight percent, or about 1to about 30 weight percent, e.g. about 10 to about 25 weight percent orabout 5 to about 20 weight percent therapeutic agent.

In an embodiment, a nanoemulsion process is provided, such as theprocess represented in FIGS. 1 and 2. For example, a therapeutic agent,a first polymer (for example, PLA-PEG or PLGA-PEG) and a second polymer(e.g. (PL(G)A or PLA), with an organic solution to form a first organicphase. Such first phase may include about 5 to about 50% weight solids,e.g about 5 to about 40% solids, or about 10 to about 30% solids, e.g.about 10%, 15%, 20% solids. The first organic phase may be combined witha first aqueous solution to form a second phase. The organic solutioncan include, for example, acetonitrile, tetrahydrofuran, ethyl acetate,isopropyl alcohol, isopropyl acetate, dimethylformamide, methylenechloride, dichloromethane, chloroform, acetone, benzyl alcohol, Tween80, Span 80,or the like, and combinations thereof. In an embodiment, theorganic phase may include benzyl alcohol, ethyl acetate, andcombinations thereof. The second phase can be between about 1 and 50weight % , e.g., 5-40 weight %, solids. The aqueous solution can bewater, optionally in combination with one or more of sodium cholate,ethyl acetate, and benzyl alcohol.

For example, the oil or organic phase may use solvent that is onlypartially miscible with the nonsolvent (water). Therefore, when mixed ata low enough ratio and/or when using water pre-saturated with theorganic solvents, the oil phase remains liquid. The oil phase may beeemulsified into an aqueous solution and, as liquid droplets, shearedinto nanoparticles using, for example, high energy dispersion systems,such as homogenizers or sonicators. The aqueous portion of the emulsion,otherwise known as the “water phase”, may be surfactant solutionconsisting of sodium cholate and pre-saturated with ethyl acetate andbenzyl alcohol.

Emulsifying the second phase to form an emulsion phase may be performedin one or two emulsification steps. For example, a primary emulsion maybe prepared, and then emulsified to form a fine emulsion. The primaryemulsion can be formed, for example, using simple mixing, a highpressure homogenizer, probe sonicator, stir bar, or a rotor statorhomogenizer. The primary emulsion may be formed into a fine emulsionthrough the use of e.g. probe sonicator or a high pressurehomogenizer_(s) e.g. by using 1, 2, 3 or more passes through ahomogenizer. For example, when a high pressure homogenizer is used, thepressure used may be about 4000 to about 8000 psi, or about 4000 toabout 5000 psi, e.g. 4000 or 5000 psi.

Either solvent evaporation or dilution may be needed to complete theextraction of the solvent and solidify the particles. For better controlover the kinetics of extraction and a more scalable process, a solventdilution via aqueous quench may be used. For example, the emulsion canbe diluted into cold water to a concentration sufficient to dissolve allof the organic solvent to form a quenched phase. Quenching may beperformed at least partially at a temperature of about 5° C. or less.For example, water used in the quenching may be at a temperature that isless that room temperature (e.g. about 0 to about 10° C., or about 0 toabout 5° C.).

In some embodiments, not all of the therapeutic agent is encapsulated inthe particles at this stage, and a drug solubilizer is added to thequenched phase to form a solubilized phase. The drug solubilizer may befor example, Tween 80, Tween 20, polyvinyl pyrrolidone, cyclodextran,sodium dodecyl sulfate, or sodium cholate. For example, Tween-80 mayadded to the quenched nanoparticle suspension to solubilize the freedrug and prevent the formation of drug crystals. In some embodiments, aratio of drug solubilizer to therapeutic agent is about 100:1 to about10:1.

The solubilized phase may be filtered to recover the nanoparticles. Forexample, ultrafiltration membranes may be used to concentrate thenanoparticle suspension and substantially eliminate organic solvent,free drug, and other processing aids (surfactants). Exemplary filtrationmay be performed using a tangential flow filtration system. For example,by using a membrane with a pore size suitable to retain nanoparticleswhile allowing solutes, micelles, and organic solvent to pass,nanoparticles can be selectively separated. Exemplary membranes withmolecular weight cut-offs of about 300-500 kDa (˜5-25 nm) may be used.

Diafiltration may be performed using a constant volume approach, meaningthe diafiltrate (cold deionized water, e.g. about 0° C. to about 5° C.,or 0 to about 10° C.) may added to the feed suspension at the same rateas the filtrate is removed from the suspension. In some embodiments,filtering may include a first filtering using a first temperature ofabout 0 to about 5° C., or 0° C. to about 10° C., and a secondtemperature of about 20° C. to about 30° C., or 15° C. to about 35° C.For example, filtering may include processing about 1 to about 6diavolumes at about 0 to about 5° C., and processing at least onediavolume (e.g. about 1 to about 3 or about 1-2 diavolumes) at about 20°C. to about 30° C.

After purifying and concentrating the nanoparticle suspension, theparticles may be passed through one, two or more sterilizing and/ordepth filters, for example, using ˜0.2 μm depth pre-filter.

In exemplary embodiment of preparing nanoparticles, an organic phase isformed composed of a mixture of a therapeutic agent, e.g., sirolimus,and polymer (homopolymer, and co-polymer). The organic phase may bemixed with an aqueous phase at approximately a 1:5 ratio (oilphase:aqueous phase) where the aqueous phase is composed of a surfactantand optionally dissolved solvent. A primary emulsion may then formed bythe combination of the two phases under simple mixing or through the useof a rotor stator homogenizer. The primary emulsion is then formed intoa fine emulsion through the use of e.g. high pressure homogenizer. Suchfine emulsion may then quenched by, e.g. addition to deionized waterunder mixing. An exemplary quench:emulsion ratio may be aboutapproximately 8.5:1. A solution of Tween (e.g., Tween 80) can then beadded to the quench to achieve e.g. approximately 2% Tween overall,which may serves to dissolve free, unencapsulated drug. Formednanoparticles may then be isolated through either centrifugation orultrafiltration/diafiltration.

Therapeutic Agents

According to the present invention, any agents including, for example,therapeutic agents (e.g. anti-cancer agents), diagnostic agents (e.g.contrast agents; radionuclides; and fluorescent, luminescent, andmagnetic moieties), prophylactic agents (e.g. vaccines), and/ornutraceutical agents (e.g. vitamins, minerals, etc.) may be delivered bythe disclosed nanoparticles. Exemplary agents to be delivered inaccordance with the present invention include, but are not limited to,small molecules (e.g. cytotoxic agents), nucleic acids (e.g., siRNA,RNAi, and microRNA agents), proteins (e.g. antibodies), peptides,lipids, carbohydrates, hormones, metals, radioactive elements andcompounds, drugs, vaccines, immunological agents, etc., and/orcombinations thereof. In some embodiments, the agent to be delivered isan agent useful in the treatment of cancer (e.g., an anti-neoplasticagent).

In a particular embodiment, the drug may be released in a controlledrelease manner from the particle and allowed to interact locally withthe particular patient site (e.g., a tumor). The term “controlledrelease” is generally meant to encompass release of a substance (e.g., adrug) at a selected site or otherwise controllable in rate, interval,and/or amount. Controlled release encompasses, but is not necessarilylimited to, substantially continuous delivery, patterned delivery (e.g.,intermittent delivery over a period of time that is interrupted byregular or irregular time intervals), and delivery of a bolus of aselected substance (e.g., as a predetermined, discrete amount if asubstance over a relatively short period of time (e.g., a few seconds orminutes)).

The active agent or drug may be a therapeutic agent such as an mTOR(mammalian target of rapamycin) inhibitor such as sirolimus (rapamycin),temsirolimus, or everolimus, a taxane or diterpene derivative such aspaclitaxel (or its derivatives such as DHA-paclitaxel or PG-paxlitaxel)or docetaxel. In another embodiment, the active agent or drug may be avinca alkaloid such as vinorelbine, vinblastine, vincristine, orvindesine.

Pharmaceutical Formulations

Nanoparticles disclosed herein may be combined with pharmaceuticalacceptable carriers to form a pharmaceutical composition. As would beappreciated by one of skill in this art, the carriers may be chosenbased on the route of administration as described below, the location ofthe target issue, the drug being delivered, the time course of deliveryof the drug, etc.

The pharmaceutical compositions and particles disclosed herein can beadministered to a patient by any means known in the art including oraland parenteral routes. The term “patient,” as used herein, refers tohumans as well as non-humans, including, for example, mammals, birds,reptiles, amphibians, and fish. For instance, the non-humans may bemammals (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, acat, a primate, or a pig). In certain embodiments parenteral routes aredesirable since they avoid contact with the digestive enzymes that arefound in the alimentary canal. According to such embodiments, inventivecompositions may be administered by injection (e.g., intravenous,subcutaneous or intramuscular, intraperitoneal injection), rectally,vaginally, topically (as by powders, creams, ointments, or drops), or byinhalation (as by sprays).

In a particular embodiment, disclosed nanoparticles may be administeredto a subject in need thereof systemically, e.g., by IV infusion orinjection.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Inone embodiment, the inventive conjugate is suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™80. The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, theencapsulated or unencapsulated conjugate is mixed with at least oneinert, pharmaceutically acceptable excipient or carrier such as sodiumcitrate or dicalcium phosphate and/or (a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectantssuch as glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, (e) solution retarding agents such as paraffin,(f) absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay, and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets, and pills, the dosage form may alsocomprise buffering agents.

Disclosed nanoparticles may be formulated in dosage unit form for easeof administration and uniformity of dosage. The expression “dosage unitform” as used herein refers to a physically discrete unit ofnanoparticle appropriate for the patient to be treated. For anynanoparticle, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. An animal model may also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. Therapeutic efficacy and toxicity ofnanoparticles can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED₅₀ (the dose istherapeutically effective in 50% of the population) and LD₅₀ (the doseis lethal to 50% of the population). The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices may be useful in some embodiments. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for human use.

In an exemplary embodiment, a pharmaceutical composition is disclosedthat includes a plurality of nanoparticles each comprising a therapeuticagent; and a pharmaceutically acceptable excipient.

In some embodiments, a composition suitable for freezing iscomtemplated, including nanoparticles disclosed herein and a solutionsuitable for freezing, e.g., a sugar (e.g. sucrose) solution is added toa nanoparticle suspension. The sucrose may e.g., act as a cryoprotectantto prevent the particles from aggregating upon freezing. For example,provided herein is a nanoparticle formulation comprising a plurality ofdisclosed nanoparticles, sucrose and water; wherein, for example, thenanoparticles/sucrose/water is are present with about5-10%/10-15%/80-90% (w/w/w).

Methods of Treatment

In some embodiments, therapeutic particles disclosed herein may be usedto treat, alleviate, ameliorate, relieve, delay onset of, inhibitprogression of, reduce severity of, and/or reduce incidence of one ormore symptoms or features of a disease, disorder, and/or condition. Forexample, disclosed therapeutic particles, that include e.g.,temsirolimus may be used to treat renal cell carcinoma. In anotherembodiment, disclosed therapeutic particles that include e.g. everolimusor temsirolimus may be used to treat kidney cancer, glioblastomamultiforme, mantle cell lymphoma, or dermal Kaposi's sarcoma.

Also contemplated here are methods of treating patients that have beensubject to organ transplantation, by administering disclosednanoparticles that e.g. include sirolimus. Other methods contemplatedherein include methods of treating patients having tuberous sclerosiscomplex, and/or autism by administering an effective amount of adisclosed nanoparticle.

Methods contemplated herein include, for example, a method of preventingor deterring neointimal hyperplasia in a blood vessel of a patient, forexample, a patient receiving a bare metal stent in a lesion of the bloodvessel, is disclosed, comprising administering a composition comprisingdisclosed therapeutic particles such as those that include sirolimus oreverolimus. Also contemplated herein are methods of treating orpreventing restenosis (e.g. in a patient receiving a stent) comprisingadministering disclosed nanoparticles having e.g. sirolimus oreverolimus to a patient.

Disclosed treatment methods may comprise administering a therapeuticallyeffective amount of the disclosed therapeutic particles to a subject inneed thereof, in such amounts and for such time as is necessary toachieve the desired result. In certain embodiments of the presentinvention a “therapeutically effective amount” is that amount effectivefor treating, alleviating, ameliorating, relieving, delaying onset of,inhibiting progression of, reducing severity of, and/or reducingincidence of one or more symptoms or features of e.g. a cancer beingtreated.

Also provided herein are therapeutic protocols that includeadministering a therapeutically effective amount of an disclosedtherapeutic particle to a healthy individual (i.e., a subject who doesnot display any symptoms of cancer and/or who has not been diagnosedwith cancer). For example, healthy individuals may be “immunized” withan inventive targeted particle prior to development of cancer and/oronset of symptoms of cancer; at risk individuals (e.g., patients whohave a family history of cancer; patients carrying one or more geneticmutations associated with development of cancer; patients having agenetic polymorphism associated with development of cancer; patientsinfected by a virus associated with development of cancer; patients withhabits and/or lifestyles associated with development of cancer; etc.)can be treated substantially contemporaneously with (e.g., within 48hours, within 24 hours, or within 12 hours of) the onset of symptoms ofcancer. Of course individuals known to have cancer may receive inventivetreatment at any time.

In other embodiments, disclosed nanoparticles may be used to inhibit thegrowth of cancer cells, e.g., prostate cancer cells. As used herein, theterm “inhibits growth of cancer cells” or “inhibiting growth of cancercells” refers to any slowing of the rate of cancer cell proliferationand/or migration, arrest of cancer cell proliferation and/or migration,or killing of cancer cells, such that the rate of cancer cell growth isreduced in comparison with the observed or predicted rate of growth ofan untreated control cancer cell. The term “inhibits growth” can alsorefer to a reduction in size or disappearance of a cancer cell or tumor,as well as to a reduction in its metastatic potential. Preferably, suchan inhibition at the cellular level may reduce the size, deter thegrowth, reduce the aggressiveness, or prevent or inhibit metastasis of acancer in a patient. Those skilled in the art can readily determine, byany of a variety of suitable indicia, whether cancer cell growth isinhibited.

Inhibition of cancer cell growth may be evidenced, for example, byarrest of cancer cells in a particular phase of the cell cycle, e.g.,arrest at the G2/M phase of the cell cycle Inhibition of cancer cellgrowth can also be evidenced by direct or indirect measurement of cancercell or tumor size. In human cancer patients, such measurementsgenerally are made using well known imaging methods such as magneticresonance imaging, computerized axial tomography and X-rays. Cancer cellgrowth can also be determined indirectly, such as by determining thelevels of circulating carcinoembryonic antigen, prostate specificantigen or other cancer-specific antigens that are correlated withcancer cell growth. Inhibition of cancer growth is also generallycorrelated with prolonged survival and/or increased health andwell-being of the subject.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Preparation of PLA-PEG

The synthesis is accomplished by ring opening polymerization ofd,l-lactide with α-hydroxy-Ω-methoxypoly(ethylene glycol) as themacro-initiator, and performed at an elevated temperature using Tin (II)2-Ethyl hexanoate as a catalyst, as shown below(PEG Mn≈5,000 Da; PLAMn≈16,000 Da; PEGPLA M_(n)≈21,000 Da)

The polymer is purified by dissolving the polymer in dichloromethane,and precipitating it in a mixture of hexane and diethyl ether. Thepolymer recovered from this step shall be dried in an oven.

Example 2 Nanoparticle Preparation—Emulsion Process

An organic phase is formed composed of a mixture of sirolimus andpolymer (homopolymer, co-polymer, and co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:5ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant and some dissolved solvent. In order to achieve high drugloading, about 30% solids in the organic phase is used.

The primary, coarse emulsion is formed by the combination of the twophases under simple mixing or through the use of a rotor statorhomogenizer. The rotor/stator yielded a homogeneous milky solution,while the stir bar produced a visibly larger coarse emulsion. It wasobserved that the stir bar method resulted in significant oil phasedroplets adhering to the side of the feed vessel, suggesting that whilethe coarse emulsion size is not a process parameter critical to quality,it should be made suitably fine in order to prevent yield loss or phaseseparation. Therefore the rotor stator is used as the standard method ofcoarse emulsion formation, although a high speed mixer may be suitableat a larger scale.

The primary emulsion is then formed into a fine emulsion through the useof a high pressure homogenizer. The size of the coarse emulsion does notsignificantly affect the particle size after successive passes (1-3)through the homogenizer. M-110-EH (FIG. 3).

Homogenizer feed pressure was found to have a significant impact onresultant particle size. On both the pneumatic and electric M-110EHhomogenizers, it was found that reducing the feed pressure also reducedthe particle size (FIG. 4). Therefore the standard operating pressureused for the M-110EH is 4000-5000 psi per interaction chamber, which isthe minimum processing pressure on the unit. The M-110EH also has theoption of one or two interaction chambers. It comes standard with arestrictive Y-chamber, in series with a less restrictive 200 μmZ-chamber. It was found that the particle size was actually reduced whenthe Y-chamber was removed and replaced with a blank chamber.Furthermore, removing the Y-chamber significantly increases the flowrate of emulsion during processing.

After 2-3 passes the particle size was not significantly reduced, andsuccessive passes can even cause a particle size increase. The resultsare summarized in FIG. 5.

The effect of scale on particle size showed surprising scale dependence.The trend shows that in the 2-10 g batch size range, larger batchesproduce smaller particles. It has been demonstrated that this scaledependence is eliminated when considering greater than 10 g scalebatches. The amount of solids used in the oil phase was about 30%. FIG.6 depicts the effect of solids concentration on particle size.

Table A summarizes the emulsification process parameters.

TABLE A Parameter Value Observation Coarse emulsion Rotor stator Coarseemulsion size does not affect final particle size, but large formationhomogenizer coarse emulsion can cause increased oil phase retention infeed vessel Homogenizer feed 4000-5000 psi per Lower pressure reducesparticle size pressure chamber Interaction 2 × 200 μm Z- 200 μmZ-chamber yields the smallest particle size, and allows for chamber(s)chamber highest homogenizer throughput Number of 2-3 passes Studies haveshown that the particle size is not significantly homogenizer passesreduced after 2 discreet passes, and size can even increase withsuccessive passes Water phase 0.1% [Sodium cholate] can effectivelyalter particle size; value is [sodium cholate] optimized for givenprocess and formulation W:O ratio 5:1 Lowest ratio without significantparticle size increase is ~5:1 [Solids] in oil phase  30% Increasedprocess efficiency, increased drug encapsulation, workable viscosity

The fine emulsion is then quenched by addition to deionized water at agiven temperature under mixing. In the quench unit operation, theemulsion is added to a cold aqueous quench under agitation. This servesto extract a significant portion of the oil phase solvents, effectivelyhardening the nanoparticles for downstream filtration. Chilling thequench significantly improved drug encapsulation. The quench:emulsionratio is approximately 5:1.

A solution of 35% (wt %) of Tween 80 is added to the quench to achieveapproximately 2% Tween 80 overall After the emulsion is quenched asolution of Tween-80 is added which acts as a drug solubilizer, allowingfor effective removal of unencapsulated drug during filtration. Table Bindicates each of the quench process parameters.

TABLE B Summary quench process parameters. Parameter Value ObservationInitial quench <5° C. Low temperature yields higher drug encapsulationtemperature [Tween-80] solution 35% Highest concentration that can beprepared and readily disperses in quench Tween-80:drug ratio 25:1Minimum amount of Tween-80 required to effectively remove unencapsulateddrug Q:E ratio  5:1 Minimum Q:E ratio while retaining high drugencapsulation Quench ≦5° C. (with Temperature which prevents significantdrug leaching during hold/processing temp current 5:1 Q:E quench holdtime and initial concentration step ratio, 25:1 Tween- 80:drug ratio)

The temperature must remain cold enough with a dilute enough suspension(low enough concentration of solvents) to remain below the T_(g) of theparticles. If the Q:E ratio is not high enough, then the higherconcentration of solvent plasticizes the particles and allows for drugleakage. Conversely, colder temperatures allow for high drugencapsulation at low Q:E ratios (to ˜3:1), making it possible to run theprocess more efficiently.

The nanoparticles are then isolated through a tangential flow filtrationprocess to concentrate the nanoparticle suspension and buffer exchangethe solvents, free drug, and drug solubilizer from the quench solutioninto water. A regenerated cellulose membrane is used with a molecularweight cutoffs (MWCO) of 300.

A constant volume diafiltration (DF) is performed to remove the quenchsolvents, free drug and Tween-80. To perform a constant-volume DF,buffer is added to the retentate vessel at the same rate the filtrate isremoved. The process parameters for the TFF operations are summarized inTable C. Crossflow rate refers to the rate of the solution flow throughthe feed channels and across the membrane. This flow provides the forceto sweep away molecules that can foul the membrane and restrict filtrateflow. The transmembrane pressure is the force that drives the permeablemolecules through the membrane.

TABLE C TFF Parameters Optimized Parameter Value Effect MembraneRegenerated No difference in performance between RC and PES, butMaterial cellulose - solvent compatibility is superior for RC. CoarseScreen Membrane Molecular Weight 300 kDa No difference in NPcharacteristics (i.e. residual Cut off tween)Increase in flux rates isseen with 500 kDa membrane but 500 kDa is not available in RC CrossflowRate 11 L/min/m² Higher crossflow rate led to higher flux Transmembrane20 psid Open channel membranes have maximum flux rates Pressure between10 and 30 psid. Coarse channel membranes have maximum flux rates withmin TMP (~20 psid). Concentration of 30 mg/ml Diafiltration is mostefficient at [NP] ~50 mg/ml with Nanoparticle open channel TFF membranesbased on flux rates and Suspension for throughput. With coarse channelmembranes the flux rate Diafiltration is optimized at ~30 mg/ml in thestarting buffer. Number of ≧15 (based on About 15 diavolumes are neededto effectively remove Diavolumes flux increase) tween-80. End point ofdiafiltration is determined by in- process control (flux increaseplateau). Membrane Area ~1 m²/kg Membranes sized based on anticipatedflux rates and volumes required.

The filtered nanoparticle slurry is then thermal cycled to an elevatedtemperature during workup. A small portion (typically 5-10%) of theencapsulated drug is released from the nanoparticles very quickly afterits first exposure to 25° C. Because of this phenomenon, batches thatare held cold during the entire workup are susceptible to free drug ordrug crystals forming during delivery or any portion of unfrozenstorage. By exposing the nanoparticle slurry to elevated temperatureduring workup, this ‘loosely encapsulated’ drug can be removed andimprove the product stability at the expense of a small drop in drugloading. 5 diavolumes is used as the amount for cold processing prior tothe 25° C. treatment.

After the filtration process the nanoparticle suspension is passedthrough a sterilizing grade filter (0.2 μm absolute). Pre-filters areused to protect the sterilizing grade filter in order to use areasonable filtration area/time for the process. Values are assummarized in Table D.

TABLE D Parameter O Value Effect Nanoparticle 50 mg/ml Yield losses arehigher at higher Suspension [NP], but the ability to filter atConcentration 50 mg/ml obviates the need to aseptically concentrateafter filtration Filtration flow ~1.3 L/min/m² Filterability decreasesas flow rate rate increases

The filtration train is Ertel Alsop Micromedia XL depth filter M953Pmembrane (0.2 μm Nominal); Pall SUPRAcap with Seitz EKSP depth filtermedia (0.1-0.3 μm Nominal); Pall Life Sciences Supor EKV 0.65/0.2 micronsterilizing grade PES filter.

0.2 m2 of filtration surface area per kg of nanoparticles for depthfilters and 1.3 m2 of filtration surface area per kg of nanoparticlesfor the sterilizing grade filters can be used.

Example 3 Cryoprotectant

Freezing a suspension of nanoemulsion nanoparticles in deionized wateralone results in particle aggregation. This is believed to be due tocrystallization and entanglement of PEG chains on the nanoparticlesurfaces. Sugar-based excipients (sucrose, trehalose, or mannitol) canact to cryoprotect these nanoparticles under freeze/thaw conditions,with a concentrations as low as 1 wt % for dilute (˜10 mg/ml)nanoparticle suspensions. One formulation includes 10 wt % sucrose,which contains excess sucrose to what is required and is the sameosmolality as physiological saline.

Table E shows that 16/5 PLA-PEG co-polymer is less susceptible tofreeze-thaw aggregation.

TABLE E Post- Original F/T Median Median Post-F/T Post-F/T PSD/ PS Poly-Baseline Description PD (nm) dispersity Index 1:1 45/5 and PLA 143.4,0.124 358.9 0.358 0.0/23.16% (baseline) 16/5 PLA-PEG and 186.7, 0.080189.5 0.126 9.7/91.57% PLA (1:1) 2:1:1 16/5:PLA:cetyl 174.1, 0.084 232.70.146 0.0/61.19% 2:1:1 45/5:PLA:cetyl 111.0, 0.182 0 0 0.0/1.55%  16/5PLA-PEG alone 218.8, 0.098 226.9 0.03 7.3/60.56% 16/5 PLA-PEG and 222.2,0.126 230.7 0.065 4.1/35.36% PLA (3:1) 45/5 PLGA-PEG and 162.7, 0.099178.6 0.091 7.7/95.41% PLA (3:1) 2:1:1 45/5 PLA- 115.9, 0.154 734.60.392 0.0/13.27% PEG:PLA:cetyl

Example 4 In Vitro Release

An in vitro release method is used to determine the initial burst phaserelease from nanoparticles at both ambient and 37° C. conditions. Inorder to maintain sink conditions and prevent nanoparticles fromentering the release samples, a dialysis system was designed. Afterobtaining an ultracentrifuge capable of pelleting 100 nm particles, thedialysis membranes were eliminated and centrifugation was used toseparate released drug from encapsulated drug.

The dialysis system is as follows: 3 mL slurry of sirolimusnanoparticles (approx 250 μg/mL sirolimus PLGA/PLA nanoparticles,corresponding to 2.5 mg/mL solid concentration) in DI-water is placedinto the inner tube of a 300 kDa MWCO dialyzer by pipetting. Thenanoparticle is suspension in this media. The dialyzer is placed into aglass bottles containing 130 ml release media (2.5% hydroxyl betacyclodextrin in PBS), which is continually stirred at 150 rpm using ashaker to prevent the formation of an unstirred water layer at themembrane/outer solution interface. At pre-determined time points,aliquot of samples (1 mL) were withdrawn from the outer solution(dialysate) and analyzed for sirolimus concentration by HPLC.

The centrifugal system is run using similar conditions at lowersuspension volumes without dialysis bags. Samples are centrifuged at60,000 g for 30 minutes and the supernatant is assayed for sirolimuscontent to measured released sirolimus.

Example 5 Particle Size Analysis

Particle size is analyzed by two techniques—dynamic light scattering(DLS) and laser diffraction. DLS is performed using a BrookhavenZetaPals instrument at 25° C. in dilute aqueous suspension using a 660nm laser scattered at 90° and analyzed using the Cumulants and NNLSmethods (TP008). Laser diffraction is performed with a Horiba LS950instrument in dilute aqueous suspension using both a HeNe laser at 633nm and an LED at 405 nm, scattered at 90° and analyzed using the Mieoptical model (TP009). The output from the DLS is associated with thehydrodynamic radius of the particles, which includes the PEG ‘corona’,while the laser diffraction instrument is more closely associated withthe geometric size of the PLA particle ‘core’.

Example 6

Nanoparticle batches were prepared using the general procedure ofExample 2, with 80% (w/w) Polymer-PEG or Polymer-PEG with homopolymerPLA at 40% (w/w) each, with a batch of % total solids of 5%, 15% and30%. Solvents used were: 21% benzyl alcohol and 79% ethyl acetate (w/w).For each 2 gram batch size, 400 mg of drug was used and 1.6 g of 16-5Polymer-PEG or 0.8 g of 16-5 Polymer-PEG+0.8 g of 10 kDa PLA(homopolymer) was used. The diblock polymer 16-5 PLA-PEG or PLGA-PEG(50:50 L:G) was used, and if used, the homopolymer: PLA with a Mn=6.5kDa, Mw=10 kDa, and Mw/Mn=1.55.

The organic phase (drug and polymer)is prepared in 2 g batches: To 20 mLscintillation vial add drug and polymer(s). The mass of solvents neededat % solids concentration is shown below:

i. 5% solids: 7.98 g benzyl alcohol+30.02 g ethyl acetate

ii. 15% solids: 2.38 g benzyl alcohol+8.95 g ethyl acetate

iii. 30% solids: 0.98 g benzyl alcohol+3.69 g ethyl acetate

An aqueous solution is prepared with 0.5% sodium cholate, 2% benzylalcohol, and 4% ethyl acetate in water. To a 2 L bottle add 7.5 g sodiumcholate, 1402.5 g of DI water, 30 g of benzyl alcohol and 60 g of ethylacetate, and mix on stir plate until dissolved.

For the formation of emulsion, a ratio of aqueous phase to oil phase of5:1 is used. The organic phase is poured into the aqueous solution andhomogenized using IKA for 10 seconds at room temperature to form courseemulsion. The solution is fed through the homogenizer (110S) at 9 Kpsi(45 psi on gauge) for 2 discreet passes to form nanoemulsion.

The emulsion is poured into quench (D.I. water) at <5 C while stirringon stir plate. Ratio of quench to emulsion is 8:1. 35% (w/w) Tween 80 isadded in water to quench at ratio of 25:1 Tween 80 to drug. Thenanoparticles are concentrated through TFF and the quench isconcentrated on TFF with 500 kDa Pall cassette (2 membrane) to ˜100 mL.Diafiltering is used using ˜20 diavolumes (2 liter) of cold DI water,and the volume is brought down to minimal volume then collect finalslurry, ˜100 mL. The solids concentration of unfiltered final slurry isdetermined by the using tared 20 mL scintillation vial and adding 4 mLfinal slurry and dry under vacuum on lyo/oven and the weight ofnanoparticles in the 4 mL of slurry dried down is determined.Concentrated sucrose (0.666 g/g) is added to final slurry sample toattain 10% sucrose.

Solids concentration of 0.45 um filtered final slurry was determined byfiltering about 5 mL of final slurry sample before addition of sucrosethrough 0.45 μm syringe filter; to tared 20 mL scintillation vial add 4mL of filtered sample and dry under vacuum on lyo/oven.

The remaining sample of unfiltered final slurry was frozen with sucrose.Rapamycin (sirolimus) formulations:

Drug Release of Drug (t = hr) Name Polymer Size (nm) Loading T = 0 T = 2T = 4 T = 24  5% Solid 16/5 PLA/PEG 123.1 3.61% ND ND ND ND 16/5PLA/PEG + PLA 119.7 4.49% ND ND ND ND 15% Solid 16/5 PLA/PEG 82.1 4.40%ND ND ND ND 16/5 PLA/PEG + PLA 120.6 11.51%  ND ND ND ND 23% Solid 16/5PLA/PEG 88.1 7.40% ND ND ND ND 16/5 PLA/PEG + PLA 118.3  7.8% ND ND NDND 30% Solid 16/5 PLA/PEG 88.5 10.26%  8.5 17.3 22.4 64.2 16/5 PLA/PEG +PLA 118.3 10.18%  9.3 30.4 44.7 98.2

The effect of solid contents and the inclusions of poly(lactic) acidhomopolymer is shown in FIG. 7.

In-vitro release experiments are studied by dispersing nanoparticles inPBS containing 10% (w/w) of Tween 20 (T20) at 37° C. T20 was used toincrease the solubility of rapamycin in PBS to levels well detectable byHPLC as well as maintaining the sink condition. 3 mL of drug-loadednanoparticles were redispersed in 130 mL of release medium in a jar at aknown concentration (approximately 250 μg/ml). These volumes were chosento ensure that the maximum concentration of the drug in the releasemedium would always be less than 10% of the maximum solubility, i.e.,sink conditions. The media and nanoparticle suspension is stirred at 150rpm. At pre-determined time points, 4 ml of aliquots were centrifuged at50,000 rpm (236,000 g) for 1 hr to separate the nanoparticles from theelution media. The elution media is injected in to a HPLC to determinedrug released from the nanoparticles. The release of rapamycin showedslow and sustained release, as shown in FIG. 8.

Example 7

Nanoparticles were prepared as in Example 2 and 6, except temsirolimuswas used with 30% solid content in the organic phase before emulsion:

Size Drug Release of Drug (t = hr) Name Lot # Polymer (nm) Loading T = 0T = 2 T = 4 T = 24 30% Solid 45-48-1 16/5 PLA/PEG 97.5 9.9% 11.5 15.617.9 40.9 45-48-2 16/5 PLA/PEG + 112.8 14.2% 9.8 22.3 29.9 88.0 PLA45-100-1 16/5 PLGA/PEG + 150.3 4.6 ND ND ND ND PLA 50-52-6 16/5PLGA/PEG + ND 6.9 10.6 35.7 45.8 87.0 PLA

FIG. 9 depicts the weight % of temsirolimus and FIG. 10 depicts thenanoparticle for the different polymeric nanoparticles havingtemsirolimus. The results of an in-vitro release experiment as inExample 6 shows the slow and sustained release of temsirolimus showedslow and sustained release, as shown in FIG. 11.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

1. A therapeutic nanoparticle having a hydrodynamic diameter of thetherapeutic nanoparticle of about 70 to about 130 nm; comprising: about5 to about 30 weight percent of a mTOR inhibitor; about 10 to about 99weight percent of a diblock poly(lactic) acid-poly(ethylene)glycolcopolymer, wherein said diblock poly(lactic) acid-poly(ethylene)glycolcopolymer comprises poly(lactic acid) having a number average molecularweight of about 15 to about 20 kDa and poly(ethylene)glycol having anumber average molecular weight of about 4 to about 6 kDa; about 0.2 toabout 10 weight percent of a polymer conjugate represented by:PLA-PEG-ligand; wherein the ligand is covalently bound to the PEG orcovalently bound to the PEG through an alkylene linker, and wherein PLAis poly(lactic)acid and PEG is poly(ethylene)glycol; and wherein thetherapeutic nanoparticle releases less than 10% of the therapeutic agentover about one minute when placed in a phosphate buffer solution at 37°C.
 2. The therapeutic nanoparticle of claim 1 wherein said mTORinhibitor is selected from the group consisting of sirolimus,temsirolimus, and everolimus, and pharmaceutically acceptable saltsthereof.
 3. The therapeutic nanoparticle of claim 1, wherein thehydrodynamic diameter is about 70 to about 120 nm.
 4. The therapeuticnanoparticle of claim 1, wherein the therapeutic nanoparticlesubstantially retains the therapeutic agent for at least 5 days at 25°C.
 5. The therapeutic nanoparticle of claim 1, comprising about 10 toabout 20 weight percent of the mTOR inhibitor.
 6. The therapeuticnanoparticle of claim 1, comprising about 40 to about 90 weight percentdiblock poly(lactic) acid-poly(ethylene)glycol copolymer.
 7. Thetherapeutic nanoparticle of claim 1, wherein the particle releases lessthan about 5% of the therapeutic agent over 1 hour when placed in aphosphate buffer solution at room temperature.
 8. The therapeuticnanoparticle of claim 1, wherein the particle releases less than about10% of the therapeutic agent over 24 hours when placed in a phosphatebuffer solution at room temperature.
 9. The therapeutic nanoparticle ofclaim 1, wherein the ligand has molecular weight of about 100 g/mol toabout 6000 g/mol.
 10. The therapeutic nanoparticle of claim 9, whereinthe ligand has a molecular weight of about 100 g/mol to about 500 g/mol.11. The therapeutic nanoparticle of claim 10, wherein the PLA-PEG-Ligandcomprises a PLA having number average molecular weight of about 10 kDato about 20 kDa and a PEG having a number average molecular weight ofabout 4 kDa to about 8 kDa.
 12. The therapeutic nanoparticle of claim 1,wherein the diblock poly(lactic) acid-poly(ethylene)glycol copolymercomprises poly(lactic acid) having a number average molecular weight ofabout 16 kDa.
 13. The therapeutic nanoparticle of claim 12, wherein saiddiblock poly(lactic) acid-poly(ethylene)glycol copolymer comprisespoly(ethylene)glycol having a number average molecular weight of about 5kDa.
 14. A method of treating lymphoma comprising administering to apatient in need thereof an effective amount of the therapeuticnanoparticle of claim
 13. 15. A pharmaceutical composition comprising: aplurality of polymeric nanoparticles each having a hydrodynamic diameterof about 60 nm to about 140 nm and comprising about 3 to about 40 weightpercent of a mTOR inhibitor; about 10 to about 99 weight percent of adiblock poly(lactic)acid-poly(ethylene)glycol copolymer comprisingpoly(lactic)acid having a number average molecular weight of about 15 toabout 20 kDa and poly(ethylene)glycol having a number average molecularweight of about 4 to about 6 kDa, about 0.2 to about 10 weight percentof a polymer conjugate represented by: PLA-PEG-ligand; wherein theligand is covalently bound to the PEG or covalently bound to the PEGthrough an alkylene linker, and wherein PLA is poly(lactic)acid and PEGis poly(ethylene)glycol and a saccharide; wherein said nanoparticles arestable for at least 3 days when held at 25° C. in said composition. 16.The pharmaceutical composition of claim 15, wherein the saccharide issucrose.